Clostridium difficile (C. difficile) is a gram-positive bacterium that causes gastrointestinal disease in humans. C. difficile is the most common cause of infectious diarrhea in hospital patients, and is one of the most common nosocomial infections overall (Kelly et al., New Eng. J. Med., 330:257-62, 1994). In fact, disease associated with this pathogen may afflict as many as three million hospitalized patients per year in the United States (McFarland et al., New Eng. J. Med., 320:204-10, 1989; Johnson et al., Lancet, 336:97-100, 1990).
Treatment with antibiotics such as ampicillin, amoxicillin, cephalosporins, and clindamycin that disrupt normal intestinal flora can allow colonization of the gut with C. difficile and lead to C. difficile disease (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998). The onset of C. difficile disease typically occurs four to nine days after antibiotic treatment begins, but can also occur after discontinuation of antibiotic therapy. C. difficile can produce symptoms ranging from mild to severe diarrhea and colitis, including pseudomembranous colitis (PMC), a severe form of colitis characterized by abdominal pain, watery diarrhea, and systemic illness (e.g., fever, nausea). Relapsing disease can occur in up to 20% of patients treated for a first episode of disease, and those who relapse are at a greater risk for additional relapses (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).
C. difficile disease is believed to be caused by the actions of two exotoxins, toxin A and toxin B, on gut epithelium. Both toxins are high molecular weight proteins (280-300 kDa) that catalyze covalent modification of Rho proteins, small GTP-binding proteins involved in actin polymerization, in host cells. Modification of Rho proteins by the toxins inactivates them, leading to depolymerization of actin filaments and cell death. Both toxins are lethal to mice when injected parenterally (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).
C. difficile disease can be diagnosed by assays that detect the presence or activity of toxin A or toxin B in stool samples, e.g., enzyme immunoassays. Cytotoxin assays can be used to detect toxin activity. To perform a cytotoxin assay, stool is filtered to remove bacteria, and the cytopathic effects of toxins on cultured cells are determined (Merz et al., J. Clin. Microbiol., 32:1142-47, 1994).
C. difficile treatment is complicated by the fact that antibiotics trigger C. difficile associated disease. Nevertheless, antibiotics are the primary treatment option at present. Antibiotics least likely to cause C. difficile associated disease such as vancomycin and metronidazole are frequently used. Vancomycin resistance evolving in other microorganisms is a cause for concern in using this antibiotic for treatment, as it is the only effective treatment for infection with other microorganisms (Gerding, Curr. Top. Microbiol. Immunol., 250:127-39, 2000). Probiotic approaches, in which a subject is administered non-pathogenic microorganisms that presumably compete for niches with the pathogenic bacteria, are also used. For example, treatment with a combination of vancomycin and Saccharomyces boulardii has been reported (McFarland et al., JAMA., 271(24):1913-8, 1994. Erratum in: JAMA, 272(7):518, 1994).
Vaccines have been developed that protect animals from lethal challenge in infectious models of disease (Torres et al., Infect. Immun. 63(12):4619-27, 1995). In addition, polyclonal antibodies have been shown to protect hamsters from disease when administered by injection or feeding (Giannasca et al., Infect. Immun. 67(2):527-38, 1999; Kink and Williams, Infect. Immun., 66(5):2018-25, 1998). Murine monoclonal antibodies have been isolated that bind to C. difficile toxins and neutralize their activities in vivo and in vitro (Corthier et al., Infect. Immun., 59(3):1192-5, 1991). There are some reports that human polyclonal antibodies containing toxin neutralizing antibodies can prevent C. difficile relapse (Salcedo et al., Gut., 41(3):366-70, 1997). Antibody response against toxin A has been correlated with disease outcome, indicating the efficacy of humoral responses in controlling infection. Individuals with robust toxin A ELISA responses had less severe disease compared to individuals with low toxin A antibody levels (Kyne et al., Lancet, 357(9251):189-93, 2001).
The individual role of toxin A and toxin B in disease pathogenesis, and the role of anti-toxin antibodies in protection from C. difficile disease are controversial and may depend on the host. In humans, the anti-toxin A antibody response has been correlated to disease outcome, suggesting a requirement for anti-toxin A response for protection. This observation is in contrast with reports of disease-causing C. difficile organisms that express only toxin B, implying that toxin B can contribute to disease in humans. These toxin A-negative strains can also cause disease in hamsters (Sambol et al., J. Infect. Dis., 183(12):1760-6, 2001).